A new approach for the characterization of the C-terniiiials of proteins has been developed. The strategy involves three steps. First, the protein is degraded enzymatically with trypsin or Lys-C. Second the C-tenninal peptide is separated from all the other proteolysis products through the use of anhydrotrypsin, a modified trypsin that binds only peptides containing K or R at the C-terminus. Finally, the C-terminal peptide is characterized by MALDI. The strategy was tested and shown to work well on a series of designed synthetic peptides as well as on the transcription factor Hairy, where the C-terminal peptide was readily isolated and identified.